Fast results hybrid capture assay and system

ABSTRACT

The present invention comprises a method that provides fast and reliable results for detecting the presence of a target nucleic acid molecule in a sample.

This application is a Continuation application of U.S. application Ser.No. 12/605,540, filed Oct. 26, 2009, which claims priority to both U.S.Provisional Patent Application No. 61/108,687, filed Oct. 27, 2008 andU.S. Provisional Application No. 61/174,848, filed May 1, 2009. Thecontents of all applications are hereby incorporated by reference intheir entireties.

FIELD

The present invention relates to methods, reagents, systems, and kitsfor determining the presence of a nucleic acid in a sample.

BACKGROUND

The detection and characterization of specific nucleic acid sequencesand sequence changes have been utilized to detect the presence of viralor bacterial nucleic acid sequences indicative of an infection, thepresence of variants or alleles of mammalian genes associated withdisease and cancers, and the identification of the source of nucleicacids found in forensic samples, as well as in paternity determinations.

For example, the RNA or DNA for many microorganisms and viruses havebeen isolated and sequenced. Nucleic acid probes have been examined fora large number of infections. Detectable nucleic acid sequences thathybridize to complementary RNA or DNA sequences in a test sample havebeen previously utilized. Detection of the probe indicates the presenceof a particular nucleic acid sequence in the test sample for which theprobe is specific. In addition to aiding scientific research, DNA or RNAprobes can be used to detect the presence of viruses and microorganismssuch as bacteria, yeast and protozoa as well as genetic mutations linkedto specific disorders in patient samples.

Nucleic acid hybridization probes have the advantages of highsensitivity and specificity over other detection methods and do notrequire a viable organism. Hybridization probes can be labeled, forexample with a radioactive substance that can be easily detected, orwith biochemical markers such as, for example, biotin, that allows fortheir capture and detection. Nucleic acid molecules may also by capturedby a first antibody that is specific to DNA hybrids, wherein the hybridsmay comprise DNA-RNA hybrids, DNA-DNA hybrids or RNA-RNA hybrids. Thehybrids may subsequently be detected by a second, labeled, antibody thatmay be, for example, labeled with a biochemical marker such as alkalinephosphatase or any other marker capable of detection.

As nucleic acid sequence data for genes from humans and pathogenicorganisms accumulates, the demand for fast, cost-effective, andeasy-to-use tests increases. There is a need to provide novel andeffective methods, compositions, and kits for determining target nucleicacids in a rapid, cost-effective, and reliable manner in geographicalareas where access to medical care is not readily available. There isalso a need to provide these assays in a rapid-screen format that can beused in developing countries. The methods and assays of the presentinvention meet these needs and may be used in manual, partiallyautomated, automated, and non-automated systems.

Clinical analysis in developing countries and geographical areas whereaccess to medical care is not readily available presents uniquechallenges. The invention described herein achieves an acceptableresolution that balances the importance of these challenges in suchcountries and areas. For instance, speed in obtaining results isparticularly important in locations where women travel great distancesto provide specimens for analysis. In such locations, it is advantageousthat results are obtained within several hours or the same day while thepatient is still present to avoid loss to follow-up associated withtraveling from home to the test site.

Other factors facing developing countries are the cost of running theassay and the instrumentation needed to run the assay. Repeat pipettorsand single pipettes are just two types of devices that are routinelyused in developed countries but are potentially cost prohibitive indeveloping countries. Accordingly, there is a need for medical devicesand products which employ cheaper and more readily accessiblealternatives in developing countries.

SUMMARY

One aspect relates to a method for determining the presence of a targetnucleic acid molecule in a sample containing biological material. Thebiological material can include a cervical epithelial cell or nucleicacid from a cervical cell. Using the disclosed methods, thedetermination of whether a target nucleic acid molecule is present in asample can be obtained relatively rapidly, for example within a periodof less than about two or three hours.

In an aspect, a method for determining the presence of a target nucleicacid molecule in a sample comprises:

-   -   a) suspending the sample in a collection medium;    -   b) releasing target nucleic acid molecules from the sample into        the collection medium;    -   c) converting double-stranded target nucleic acid molecules to        single-stranded target nucleic acid molecules;    -   d) contacting one or more probes with the single-stranded target        nucleic acid molecules under conditions that allow the probes        and target single-stranded target nucleic acid molecules to        hybridize forming double-stranded nucleic acid hybrids;    -   e) capturing the double-stranded nucleic acid hybrids;    -   f) separating the double-stranded nucleic acid hybrids from        un-bound single-stranded target nucleic acid molecules; and    -   g) detecting the double-stranded nucleic acid hybrids, thereby        indicating the presence of the target nucleic acid.

In one aspect, the method may be predominantly manual, requiring humaninput. Another aspect relates to the rapid detection of target nucleicacid molecules in a sample. The detection method may be automated,either fully automated, or partially automated—in other words requiringsome human input.

Another aspect relates to the detection of target nucleic acid moleculesin multiple samples at the same time or within a very short period oftime, for example in a machine or a series of machines.

Yet another aspect relates to an instrument for running a method for thedetection of a target nucleic acid molecule in a simple footprint. Theinstrument combines many, or all, of other individual instruments thatperform the steps of the method.

Another aspect relates to a portable system for evaluating the detectionof a target nucleic acid molecule in a sample.

Another aspect relates to a kit for the detection of a target nucleicacid molecule in a sample.

A further aspect relates to reagents within a collection medium intowhich a sample containing a target nucleic acid molecule are collected.The target nucleic acid molecule can be kept in the collection mediumwith minimal degradation of the target nucleic acid molecule over a timeperiod of weeks or months. In an aspect, DNA-based target samplematerial can be kept in the collection medium with minimal degradationof the target nucleic acid molecule over a time period of weeks ormonths. In an aspect the detergent-based collection medium allows forthe rapid analysis and processing of a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that the detergent-based collection medium holds magneticbeads in microtiter plate wells better than known collection or sampletransport medium (STM) (non-detergent based medium).

FIG. 2 shows that samples having only 0.2 pg target nucleic acid (DNA)per ml of sample provide a readable signal using methods of the presentinvention.

FIG. 3 shows clinical specimen stability at room temperature for 21days.

FIG. 4 shows clinical specimen stability at 33° C. for 21 days.

FIG. 5 shows test results demonstrate an S/N≧2.0 for a 0.2 pg/ml HPV 16plasmid which is equivalent to 1000 copies of HPV 16 DNA.

FIG. 6 shows a system for detecting the presence of a target nucleicmolecule acid in a sample including a heater configured for heatingmultiple samples; a luminometer; and a monitor.

FIG. 7 shows different reagents associated with the detection assay. Thereagent vials are color coded for ease of use and can be included in akit.

FIG. 8 shows a monitor used in conjunction with the system for detectingthe presence of nucleic acid molecules.

FIG. 9 shows 11 day stability data for soft pellets suspended in adetergent-based collection medium and stored at room temperature.

DETAILED DESCRIPTION

The present disclosure covers methods, compositions, reagents, systems,and kits for rapidly determining the presence of a nucleic acid moleculein a sample. The methods, compositions, reagents, systems, and kits maybe used for clinical diagnostic purposes, including but not limited tothe detection and identification of pathogenic organisms and thedetection of a genetic predisposition to a particular disease.

In one aspect, the present disclosure provides a method for determiningthe presence of a target nucleic acid molecule in a sample. The methodcomprises:

-   -   a) suspending the sample in a collection medium comprising a        detergent;    -   b) denaturing the target nucleic acid molecule;    -   c) contacting one or more polynucleotide probes with the target        nucleic acid molecule under conditions that allow the probes and        the target nucleic acid molecule to hybridize, thereby forming a        double-stranded nucleic acid hybrid;    -   d) capturing the double-stranded nucleic acid hybrid on a solid        support coated with a first antibody specific for the        double-stranded hybrid nucleic acid hybrid, thereby forming a        double-stranded nucleic acid hybrid/solid support complex;    -   e) separating the double-stranded nucleic acid hybrid/solid        support complex from unbound nucleic acid;    -   f) conjugating the complex with a second antibody that is        specific for either the double-stranded nucleic acid hybrid or        specific for the first antibody to form a double-stranded        nucleic acid hybrid/solid support antibody complex; wherein the        second antibody is labeled with a detectable marker;    -   g) washing the double-stranded nucleic acid hybrid/solid support        antibody complex with a wash buffer comprising a detergent; and    -   h) detecting the label on the second antibody wherein the        detecting indicates the presence of the target nucleic acid        molecule.

In another aspect, the present disclosure provides a method fordetermining the presence of a target nucleic acid molecule in a sampleincluding suspending a sample in a collection medium including adetergent; denaturing a target nucleic acid molecule; contacting one ormore polynucleotide probes with the target nucleic acid molecule underconditions that allow the probes and the target nucleic acid molecule tohybridize or bind, and capturing the double-stranded nucleic acid hybridon a solid support coated with a first antibody specific for thedouble-stranded hybrid nucleic acid hybrid.

In an aspect, the present disclosure provides a method for determiningthe presence of a target nucleic acid molecule in a sample includingsuspending a sample in a collection medium including a detergent;denaturing a target nucleic acid molecule; contacting one or morepolynucleotide probes with the target nucleic acid molecule underconditions that allow the probes and the target nucleic acid molecule tohybridize or bind, capturing the double-stranded nucleic acid hybrid ona solid support coated with a first antibody specific for thedouble-stranded hybrid nucleic acid hybrid and separating thedouble-stranded nucleic acid hybrid/solid support complex from unboundnucleic acid.

In an aspect, the present disclosure provides a method for determiningthe presence of a target nucleic acid molecule in a sample includingsuspending a sample in a collection medium including a detergent;denaturing a target nucleic acid molecule; contacting one or morepolynucleotide probes with the target nucleic acid molecule underconditions that allow the probes and the target nucleic acid molecule tohybridize or bind, capturing the double-stranded nucleic acid hybrid ona solid support coated with a first antibody specific for thedouble-stranded hybrid nucleic acid hybrid, thereby forming adouble-stranded nucleic acid hybrid/solid support complex; separatingthe double-stranded nucleic acid hybrid/solid support complex fromunbound nucleic acid; and conjugating the complex with a second antibodythat is specific for either the double-stranded nucleic acid hybrid orspecific for the first antibody to form a double-stranded nucleic acidhybrid/solid support antibody complex.

In another aspect, the present disclosure provides a method fordetermining the presence of a target nucleic acid molecule in a sampleincluding suspending a sample in a collection medium including adetergent; denaturing a target nucleic acid molecule; contacting one ormore polynucleotide probes with the target nucleic acid molecule underconditions that allow the probes and the target nucleic acid molecule tohybridize or bind, capturing the double-stranded nucleic acid hybrid ona solid support coated with a first antibody specific for thedouble-stranded hybrid nucleic acid hybrid, thereby forming adouble-stranded nucleic acid hybrid/solid support complex; andseparating the double-stranded nucleic acid hybrid/solid support complexfrom unbound nucleic acid; conjugating the complex with a secondantibody that is specific for either the double-stranded nucleic acidhybrid or specific for the first antibody to form a double-strandednucleic acid hybrid/solid support antibody complex; wherein the secondantibody is labeled with a detectable marker; and washing thedouble-stranded nucleic acid hybrid/solid support antibody complex witha wash buffer comprising a detergent.

In another aspect, the present disclosure provides a method fordetermining the presence of a target nucleic acid molecule in a sample,the method comprising:

-   -   a) suspending the sample in a collection medium comprising a        detergent;    -   b) denaturing the target nucleic acid molecule in the sample;    -   c) forming a double-stranded nucleic acid hybrid by contacting        at least one polynucleotide probe with the target nucleic acid        molecule;    -   d) forming a double-stranded nucleic acid hybrid-support complex        by capturing the double-stranded nucleic acid hybrid on a        support, wherein the support comprises a first antibody;    -   e) forming a double-stranded nucleic acid hybrid-support-second        antibody complex by contacting the double-stranded nucleic acid        hybrid-support complex with a second antibody, wherein the        second antibody is labeled with a detectable marker;    -   f) washing the double-stranded nucleic acid        hybrid-support-second antibody complex with a wash buffer; and    -   g) detecting the marker on the second antibody wherein the        detecting indicates the presence of the target nucleic acid        molecule.

In one aspect, the solid support comprises a modified paramagnetic beadthat is coated or has attached thereto a first antibody immunospecificfor double-stranded hybrid nucleic acids. A magnetic field is used toseparate the double-stranded nucleic acid-magnetic bead-antibody complexfrom non-bound nucleic acid.

In an aspect, the method does not include a sample pre-treatment step.For example, the detergent-based collection medium allows for reducedsample preparation time which, in turn, can lead to accelerateddetection of target nucleic acid molecules. The sample can be analyzedby methods, assays, or the apparatus of the disclosure in adirect-to-assay manner. In an example, purification steps are notperformed on the sample prior to evaluation using assays of thedisclosure. In an aspect, crude lysate is directly analyzed by themethods, assays, or the apparatus of the disclosure. In another aspect,the sample does not undergo a target amplification step.

One aspect relates to a method of diagnosing cancer by utilizingmethods, kits, assays, and the apparatus provided herein. In one aspect,cervical cancer is detected by identifying nucleic acid moleculesassociated with HPV and HPV variants. In another aspect, cervicalintraepithelial neoplasia (CIN) can be screened for using methods, kits,assays, and the apparatus provided herein. The detected cancer can besubsequently treated after being diagnosis by the methods, kits, assays,and the apparatus provided herein. In an aspect, the diagnosed cancer iscervical cancer and variants thereof.

In one aspect, the disclosure provides a composition comprising abiological sample suspended in a collection medium comprising about 0.5%to about 2.0% NP-40, about 0.10% to about 0.40% sodium deoxycholate,about 25 mM to about 75 mM Tris-HCl, about 10 mM to about 50 mM EDTA,about 50 mM to about 200 mM NaC1, and about 0.01% to about 0.10% sodiumazide.

In an aspect, the disclosure provides for a composition comprising

-   -   (a) a biological sample suspended in about 0.5% to about 2.0%        NP-40, about 0.10% to about 0.40% sodium deoxycholate, about 25        mM to about 75 mM Tris-HCl, about 10 mM to about 50 mM EDTA,        about 50 mM to about 200 mM NaC1, and about 0.01% to about 0.10%        sodium azide; and    -   (b) at least one or more polynucleotide probes.

In an aspect, the disclosure provides for a composition comprising

-   -   (a) a biological sample suspended in a collection medium        comprising about 0.5% to about 2.0% NP-40, about 0.10% to about        0.40% sodium deoxycholate, about 25 mM to about 75 mM Tris-HC1,        about 10 mM to about 50 mM EDTA, about 50 mM to about 200 mM        NaCl, and about 0.01% to about 0.10% sodium azide;    -   (b) at least one or more polynucleotide probes; and    -   (c) a first antibody.

In an aspect, the disclosure provides for a composition comprising

-   -   (a) a biological sample suspended in a collection medium        comprising about 0.5% to about 2.0% NP-40, about 0.10% to about        0.40% sodium deoxycholate, about 25 mM to about 75 mM Tris-HCl,        about 10 mM to about 50 mM EDTA, about 50 mM to about 200 mM        NaCl, and about 0.01% to about 0.10% sodium azide;    -   (b) a first antibody; and    -   (c) a second antibody.

In an aspect, the disclosure provides for a composition comprising

-   -   (a) a biological sample suspended in a collection medium        comprising about 0.5% to about 2.0% NP-40, about 0.10% to about        0.40% sodium deoxycholate, about 25 mM to about 75mM Tris-HCl,        about 10 mM to about 50 mM EDTA, about 50 mM to about 200 mM        NaCl, and about 0.01% to about 0.10% sodium azide;    -   (b) at least or one or more polynucleotide probes;    -   (c) a first antibody; and    -   (d) a second antibody.

In an aspect, the disclosure provides for a composition comprising

-   -   (a) a biological sample suspended in a collection medium,        wherein the collection medium comprises at least one detergent;    -   (b) a denaturation reagent;    -   (c) at least one polynucleotide probe capable of binding to a        target nucleic acid molecule;    -   (d) a support coated with a first antibody; and    -   (e) a second antibody labeled with a detectable marker.

In an aspect, any of the above compositions may be used may be used withany of the collection mediums described herein.

In an aspect, the biological sample in the above compositions is acervical cell sample or a human cervical cell sample. In another aspect,the nucleic acid molecules in the biological sample are denatured. Thebiological sample in the above compositions can exhibit stability whenstored in the collection medium for at least 21 days at 33° C. In anaspect, the second antibody is labeled with a detectable marker.

Biological Sample

Methods of the present invention may be used to detect the presence of atarget nucleic acid molecule from samples, including, withoutlimitation, a specimen or culture (e.g., cellular, microbiological andviral cultures) including biological and environmental samples.Biological samples may be from an animal, including a human, fluid,solid (e.g., stool) or tissue, as well as liquid and solid food and feedproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Environmental samples include environmentalmaterial such as surface matter, soil, water and industrial samples, aswell as samples obtained from food and dairy processing instruments,apparatus, equipment, utensils, disposable and non-disposable items.

Particularly preferred are biological samples including, but not limitedto, cervical epithelial cells (e.g., a sample obtained from a cervicalswab), adenoid cells, anal epithelial cells, blood, saliva, cerebralspinal fluid, pleural fluid, milk, lymph, sputum and semen. The samplemay comprise a double-stranded nucleic acid molecule or may comprise asingle-stranded nucleic acid molecule. If a double-stranded nucleic acidmolecule is present, it may be prepared for hybridization analysis by avariety of methods known in the art, e.g., using alkali, usingproteinase K/SDS, chaotropic salts. The process of preparing adouble-stranded nucleic acid molecule for hybridization analysisgenerally involves converting it into a single-stranded nucleic acidmolecule. This process is generally known as denaturation. However, itis also contemplated that a double-stranded nucleic acid molecule may bedetected without denaturation, e.g., through a triple-strandedconstruct.

The target nucleic acid molecule in a sample can be DNA or RNA or bothDNA and RNA. The target nucleic acid molecule can be contained within alarger nucleic acid molecule. Detection of either the target nucleicacid molecule or the larger nucleic acid molecule containing the targetnucleic acid molecule is contemplated by this disclosure.

The biological sample may comprise cervical cells, especially humancervical cells. The sample can be collected with any method or deviceknown in the art, including a chemically inert collection device such asa DACRON tipped swab. Other acceptable collection devices may be usedincluding, but not limited to, cotton swab, cervical brush, flocked swab(a swab shaped like a DACRON swab but made with nylon fibers enablingcollection of more cells and easier release of cells), cervical broom,mini broom, lavage, or any collection device often used in Pap smeartesting.

In an aspect, the methods include collecting a sample from a woman over30 years of age. The method can also include collecting a sample from awoman over 30 years via a Pap smear or comparable test. The samplecollected by the Pap smear or comparable test can be a cervical cellsample.

Once the sample is collected, it may be placed in a sample tube. Thetube can be sealed to prevent contamination. The collection device(swab, brush, etc.) may further contain a mechanism by which it can bemoved once it is inside the sample tube. In one aspect, the collectiondevice contains an insert that can be moved using a magnet. In oneaspect, this insert comprises a metal. In another aspect, this insertcomprises a magnetic material. Magnetic material includes paramagnetic,ferromagnetic, and diamagnetic materials. One advantage of moving thecollection device once it is inside the sample tube is to avoid thecollection device from making contact with any sample extraction orsample detection devices. Examples of a sample extraction device includepipettes, pipette tips, dropper bottles or other low tech extractiondevices. Examples of sample detection devices include probes and probetips.

The speed of this assay is also beneficial in screening samples frompatients in remote living areas. Often patients will travel quite adistance to visit the doctor or clinic and will not likely return forsome time thereafter. Thus, it is desirable to be able to test thepatient and provide results while the patient waits at the clinic. Insome circumstances, tracking down the patient to provide test resultsand/or treat the patient after they have left the doctor's office may bedifficult. Thus, the described assays provide results over a short time,for example, between about 2 hours to about 3 hours, between about 2hours to about 4 hours, between about 3 hours to about 5 hours, betweenabout 4 hours to about 8 hours, or between about 6 hours to about 12hours. In another aspect, the described assays provide results in lessthan about 2 hours, less than about 2.5 hours, less than about 3 hours,less than about 3.5 hours, less than about 4 hours, less than about 4.5hours, less than about 5 hours, less than about 8 hours, less than about12 hours, and less than about 24 hours. Such a short turnaround timeallows the doctor to provide the patient with the results and/ortreatment the same day the patient is at the clinic.

Sample Tube

Any type of sample tube may be used. Advantageously, the sample tube maybe closed or sealed to minimize contamination. The closure may bepermanent or removable. Examples of removable closures include snapcaps, screw caps, rubber septa, foil, and film. The closure may containone or more openings or perforations, which when pierced may bere-sealable. One advantage of a closure that contains such openings orperforations is that the closure is not rendered ineffective whenpierced by, for example, a sample extraction device or sample detectiondevice. Once the sample extraction device or sample detection device isremoved, the closure re-seals, thereby minimizing contamination.

Storage of the Biological Sample

Once the sample is in the sample tube, the sample may be stored bydrying it with a substrate, or in a preservative medium, or both.Desiccation is accomplished by pressure drying or drying with chemicals.This removes most of the water and is suitable for long-term stability.Alternatively, the sample may be lyophilized (freeze-dried) with asubstrate like trehalose to ensure stability of the sample.

Another possibility is that the sample may be stored by suspending in apreservative medium, known and apparent to one of skill in the art. Thepurpose of the preservative medium is to protect biological componentsthat can degrade. For instance, the sample cells, the probe mixture, theantibody: bead complex used in the capture step, and the secondaryantibody used in the detection step are all susceptible to degradation.A preservative medium at the initial step of collection ideally providessample stability and integrity and can affect downstream steps in theprocess of nucleic acid capture and detection. In an aspect, the samplemay be stored at room temperature, refrigerated, or frozen either priorto or after the addition of the preservative medium.

Collection Medium

In an aspect, the sample may be collected and stored in a collectionmedium. The collection medium has several functions including as apreservative medium to preserve nucleic acids and inhibit nucleases toprevent degradation of nucleic acids prior to analysis. In one aspect,the collection medium contains at least one detergent. In anotheraspect, the collection medium contains at least two detergents, at leastthree detergents, or at least four detergents. In an aspect, each of thedetergents is different. In another aspect, the detergent-basedcollection medium comprises two different detergents, one which is ableto control background signal and another detergent that improvesmagnetic bead behavior, for example, migration through a viscous sample.Because the detergent improves the bead behavior, the beads can bewashed with a variety of less precise devices, such as a squirt bottle,and dropper bottle, without causing too much disruption of the beads.Such methodology may be advantageous for use in developing countrieswhere funds or access to technologically advanced equipment, such aspipetting devices, may not be available.

Further, the present invention provides a robust assay that can supportusing less precise reagent delivery devices. The reagents in this assaywere designed to be robust and withstand variability in reagent volumedelivery. For example, the neutralization step of the assay utilizes ahighly buffered pH neutral solution to bring the very basic pH of thereaction to the appropriate pH range for hybridization with very largevolume tolerances.

The use of a detergent-based collection medium for use in the assay caninclude one or more detergents. In an aspect, heat is employed duringthe hybridization, capture, and detection steps of the assay. Even withdetergent and the application of heat, antibodies used in the assayremain functional.

FIG. 1 demonstrates that a detergent-based collection medium greatlyimproves the ability to prevent magnetic bead loss and migration duringhandling as compared to standard collection mediums. The detergent-basedcollection medium may comprise, consist essentially of, or consist ofone, two, three, or four or more detergents. Detergents are known in theart and may include, but are not limited to, cationic detergents such asbut not limited to cetyl pyridinium bromide, cetyltrimethylammoniumbromide (collectively known as cetrimonium compounds) andalkylbenzyldimethylammonium chlorides (collectively known asbenzalkonium compounds), and alkyl-trimethyl-ammonium salts; anionicdetergents such as, but not limited to, sodium dodecyl sulfate (SDS),and Sarkosyl; and non-denaturing detergents such as NP-40; and otherdetergents. NP-40 is also known as Tergitol-type NP-40, which is nonylphenoxylpolyethoxylethanol. NP-40 is not powerful enough to break thenuclear membrane, but can break the plasma membrane. As such, it can beused to obtain the cytoplasmic contents of a cellular culture.

Other detergents and combination of detergents may be used, andadvantageously their combination provides the ability to controlbackground noise and improve magnetic bead behavior (when the solidsupport employed comprises magnetic beads). In certain aspects, onedetergent is an anionic detergent and the second detergent is anonanionic detergent. For example, in one aspect, the combination ofnon-ionic and anionic detergents helps to maintain low-background noise.In an aspect, a detergent-based collection medium comprises an anionicdetergent such as sodium deoxycholate, which controls background noiseand NP-40, which improves magnetic bead behavior.

The combination of these two types of detergents provides synergisticbenefits beyond a simple combination of adding two detergents together:control of background noise, better bead behavior, and increased assayspeed. The presence of these detergents (in the detergent-basedcollection medium) provides the ability to achieve faster assay results,but does not negatively impact the nucleic acid or capture antibodyduring downstream analytical steps.

In addition, the detergent-based collection medium improves removal ofthe specimen from the collection device as the sample is dissolved moreeasily. In addition, the detergent-based collection medium improves thehomogeneity of the sample compared with other collection media such asbut not limited to PRESERVCYT (uses a 40% methanol solution), STM (usesa chaotropic agent), and alcohol. The detergent-based collection mediumalso reduced sample viscosity after mixing (either manual or automated).

The concentration of NP-40 in the collection medium can range from about0.5% to about 2.0%, from about 0.1% to about 1.0%, as well as any numberwithin the recited ranges. In certain aspects, the NP-40 is present at aconcentration from about 0.8% to about 1.5%; from about 0.9% to about1.2% and in certain aspects is about 1.0%. In another aspect, the NP-40is present at a concentration from about 0.1%, about 0.2%, about 0.3%,about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%,about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%,about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%. Theconcentration of sodium deoxycholate in the collection medium can rangefrom about 0.10% to about 0.40%, from about 0.20% to about 0.30%, aswell as any number within the recited ranges. In one aspect, theconcentration of sodium deoxycholate is about 0.10%, about 0.15%, about0.20%, about 0.25%, about 0.30%, about 0.35%, or about 0.40%.

The detergent-based collection medium may comprise, consist essentiallyof, or consist of a buffer, two detergents, a chelator and apreservative. The buffer may be Tris-HCl in a concentration of fromabout 25 mM to about 75 mM; from about 30 mM to about 60 mM; from about40 mM to about 50 mM, and from about 45 mM to about 55 mM as well as anynumber within the recited ranges. The buffer may also be Tris-HCl in aconcentration of about 25 mM, about 30 mM, about 35 mM, about 40 mM,about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about70 mM, or about 75 mM.

Any preservative can be used and the choice can depend on factors suchas desired functionality, minimization side-effects, cost, etc. Suitablepreservatives include gentomycin, ProClin, dimersol, and sodium azide.The concentration of the preservatives in the collection medium dependson factors such as the type of preservative, its efficacy, itsside-effects, etc. For example, for sodium azide, the concentration ofsodium azide can range from about 0.01% to about 0.1%, from about 0.025%to about 0.075%, and from about 0.04% to about 0.06%, as well as anynumber within the recited ranges. The preservative, for example, sodiumazide, can also be present at about 0.01%, about 0.02%, about 0.03%,about 0.04%, 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%,or about 0.10%.

In one aspect the detergent-based collection medium comprises, consistsessentially of, or consists of 1.0% NP-40, 0.25% sodium deoxycholate, 50mM Tris-HCl, 25 mM EDTA, 150 mM NaCl and 0.05% sodium azide. In anotheraspect the detergent-based collection medium comprises, consistsessentially of, or consists of about 0.5% to about 2.0% NP-40, about0.10% to about 0.40% sodium deoxycholate, about 25 mM to about 75 mMTris-HCl, about 10 mM to about 50 mM EDTA, about 50 mM to about 200 mMNaCl, and about 0.01% to about 0.10% sodium azide. In other aspects thedetergent-based collection medium comprises, consists essentially of, orconsists of about 0.8% to about 1.5% NP-40, about 0.20% to about 0.40%sodium deoxycholate, about 30 mM to about 60 mM Tris-HCl, about 20 mM toabout 40 mM EDTA, about 100 mM to about 200 mM NaC1, and about 0.025% toabout 0.075% sodium azide. In yet another aspect the detergent-basedcollection medium comprises, consists essentially of, or consists ofabout 0.9% to about 1.2% NP-40, about 0.20% to about 0.30% sodiumdeoxycholate, about 30 mM to about 60 mM Tris-HCl, about 20 mM to about30 mM EDTA, about 100 mM to about 150 mM NaCl, and about 0.04% to about0.06% sodium azide.

In an aspect, the collection medium comprises, consists essentially of,or consists of NP-40 and EDTA. In another aspect, the collection mediumcomprises, consists essentially of, or consists of NP-40, EDTA, andsodium azide. In one aspect, the collection medium comprises, consistsessentially of, or consists of sodium deoxycholate, EDTA, and sodiumazide. In an aspect, the collection medium comprises, consistsessentially of, or consists of about NP-40, sodium deoxycholate, EDTA,and sodium azide. In an aspect, the collection medium comprises,consists essentially of, or consists of NP-40, sodium deoxycholate,Tris-HCl, EDTA, and sodium azide.

In another aspect, the collection medium comprises, consists essentiallyof, or consists of 0.5% to about 2.0% NP-40 and 10 mM to about 50 mMEDTA. In another aspect, the collection medium comprises, consistsessentially of, or consists of 0.5% to about 2.0% NP-40, 10 mM to about50 mM EDTA, and about 0.01% to about 0.10% sodium azide. In one aspect,the collection medium comprises, consists essentially of, or consists ofabout 0.10% to about 0.40% sodium deoxycholate, 10 mM to about 50 mMEDTA, and about 0.01% to about 0.10% sodium azide. In an aspect, thecollection medium comprises, consists essentially of, or consists ofabout 0.5% to about 2.0% NP-40, about 0.10% to about 0.40% sodiumdeoxycholate, 10 mM to about 50 mM EDTA, and about 0.01% to about 0.10%sodium azide. In an aspect, the collection medium comprises, consistsessentially of, or consists of about 0.5% to about 2.0% NP-40, about0.10% to about 0.40% sodium deoxycholate, about 25 mM to about 75 mMTris-HCl, about 10 mM to about 50 mM EDTA, and about 0.01% to about0.10% sodium azide.

In an aspect, the collection medium is a non-chaotropic medium. That is,for example, the collection medium does not include a chaotropic mediumor chaotropic salts. Without being limited, in an aspect, the collectionmedium does not include guanidine hydrochloride or urea. A potentialadvantage of using a non-chaoptropic collection medium is betterresuspension of a sample, more reproducible testing, and more uniformtesting aliquots relative to a medium which includes a chaotropic mediumor chaotropic salts.

An advantage of using a detergent-based collection medium is that itpreserves the stability of the sample. A sample stored in adetergent-based collection medium as disclosed is stable for at least 31days, and, when held at temperatures from 15° C. to 33° C. is stable forat least 21 days. In an aspect, a sample is stable when frozen in adetergent-based collection medium at −20° C. for at least six months. Inanother aspect, a cervical cell sample is stable for at least 31 days,for at least 21 days when held at temperatures from 15° C. to 33° C.,and for at least 6 months in a detergent-based collection medium at −20°C.

In an aspect, the detergent-based collection medium exhibits asensitivity for detecting cervical intraepithelial neoplasia or cancerof at least 80%, at least 90%, or at least 95% for a cut-off ratio of0.5 relative light units. In another aspect, the detergent-basedcollection medium exhibits a specificity for detecting cervicalintraepithelial neoplasia or cancer of at least 80%, at least 90%, or atleast 95% for a cut-off ratio of 0.5 relative light units. In yetanother aspect, the detergent-based collection medium exhibits asensitivity for detecting severe or moderate cervical intraepithelialneoplasia or cancer (CIN2+) of about 90% and a of specificity of about84% for a cut-off ratio of 0.5 relative light units . In an aspect, thedetergent-based medium includes 0.5% to about 2.0% NP-40, about 0.10% toabout 0.40% sodium deoxycholate, about 25 mM to about 75 mM Tris-HC1,about 10 mM to about 50 mM EDTA, about 50 mM to about 200 mM NaCl, andabout 0.01% to about 0.10% sodium azide.

A detergent-based collection medium also leads to improved assayperformance under rigorous hybridization and capture conditions (forexample, at temperatures between 65°-75° relative to collection mediumcontaining a denaturant.

The presence of one, two, three, four or more detergents can reducesample viscosity, which aids in the removal of the liquid phase from themagnetic beads, as well as aids in the mixing of samples.

In one aspect, a sample such as blood or an exfoliated cervical cellspecimen can be collected and suspended in a detergent-based collectionmedium. The sample can be is collected with a chemically inertcollection device such as a DACRON tipped swab. Any other suitable swabmay be used such as nylon fiber swabs. The sample may be stored in adetergent-based collection medium, to prevent degradation of nucleicacids prior to analysis and to maintain stability of the sample.

Samples may be collected in other known collection mediums and then canbe used in the methods described herein. Examples of other collectionmedia include PRESERVCYT, SUREPATH, DCM (DIGENE Collection Medium), andSTM (Sample/Specimen Transport Medium). Certain collection media arenucleic acid specific. For example DCM is not used when the targetnucleic acid is RNA. Samples collected in some of these media mayrequire processing before the nucleic acids in the samples can bedetected and analyzed. Various methods of processing samples (also knownas preparing the samples) are known in the art. For example, cervicalcell samples collected for cytological analysis in medium such asPRESERVCYT may be combined with a detergent-based lysis buffer followedby the addition of magnetic beads comprising nucleic acid bindingsurfaces. In addition, other cell samples collected in other knowncommonly available collection mediums may be combined with adetergent-based lysis buffer followed by the addition of magnetic beadscomprising nucleic acid binding surfaces.

Target Nucleic Acid Molecules

The target nucleic acid molecules include, without limitation, nucleicacid molecules found in specimens or cultures (e.g., cellular,microbiological and viral cultures) including biological andenvironmental samples. The target nucleic acid molecules may be found inbiological samples from an animal, including a human, fluid, solid(e.g., stool) or tissue, as well as liquid and solid food and feedproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Target nucleic acid molecules may be found inenvironmental samples and include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable and non-disposable items.

The target nucleic acid molecules found in biological samples include,but not limited to cervical samples (e.g., a sample obtained from acervical swab) or cervical cell samples, adenoid cells, anal epithelialcells, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph,sputum, urine and semen. The target nucleic acid molecules may be fromother viral, bacteria, mycobacteria or plasmodia, for examplecytomegalovirus (CMV), herpes, HIV, H1N1, chlamydia, gonorrhea,Trichomonas vaginalis, Staphylococcus aureus, tuberculosis,SARS-associated coronavirus or influenza. In an aspect the targetnucleic acid molecules are at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 98%, at least 99%, or 100% identical to nucleic acid moleculesassociated with any one of cervical samples (e.g., a sample obtainedfrom a cervical swab) or cervical cell samples, adenoid cells, analepithelial cells, blood, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum, urine and semen, other viral, bacteria,mycobacteria or plasmodia, for example cytomegalovirus (CMV), herpes,HIV, H1N1, chlamydia, gonorrhea, Neisseria gonorrhoeae (GC), Chlamydiatrachomatis (CT), Trichomonas vaginalis, Staphylococcus aureus,tuberculosis, SARS-associated coronavirus or influenza.

In one aspect, the target nucleic acid molecules are humanpapillomavirus (HPV) and include genetic variants of HPV. A variantincludes polymorphisms, mutants, derivatives, modified, altered, or thelike forms of the target nucleic acid. In one aspect, the target nucleicacid is an HPV nucleic acid. In another aspect, the HPV nucleic acid isHPV DNA of a high risk HPV type. In another aspect, the HPV nucleic acidis HPV RNA of a high risk HPV type. In another aspect the target nucleicacids are any one of high risk HPV types 16, 18, 26, 31, 33, 35, 39, 45,51, 52, 56, 58, 59, 66, 68, and 82 or any one of low risk HPV types 6,11, 40, 43, 53, 61, 67, 69, 70, 71, 72, 81, and 83.

In another aspect, the target nucleic acid molecule is at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 98%, at least 99%, or 100% identicalto nucleic acid molecules associated with any one of HPV, geneticvariants of HPV, HPV DNA of a high risk HPV type, or HPV RNA of a highrisk HPV type. In another aspect the target nucleic acids are at least70%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100%identical to nucleic acid molecules associated with any one of high riskHPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68,and 82 or any one of low risk HPV types 6, 11, 40, 43, 53, 61, 67, 69,70, 71, 72, 81, and 83.

Using methods of the present inventions, the target nucleic acidmolecule may be present at concentrations less than about 1 pg per ml,less than about 0.75 pg per ml, less than 0.5 pg per ml, less than 0.25pg per ml, and even as low as 0.2 pg per ml. As seen in FIG. 2 anexcellent signal to noise ratio is obtained when HPV-16 DNA was used asthe target nucleic acid molecule present at a concentration of 0.2 pgper ml.

As noted previously, the target nucleic acid molecule may be DNA or RNA.When the target nucleic acid molecule is DNA, the probe is preferablyRNA and when the target nucleic acid is RNA, the probe is preferablyDNA. However, a DNA probe can be used with DNA target nucleic acidmolecule and an RNA probe can be used with RNA target nucleic acidmolecule. Also as indicated previously, the target nucleic acid moleculemay determine the collection medium used.

Denaturation

After the sample is collected in a detergent-based collection medium asdescribed above, the sample may be treated with a denaturation reagentto render the target nucleic acid molecule accessible to hybridization.In one aspect, the sample is denatured with an alkaline solution. Anyalkali that can bring a solution pH to about pH 12, about pH 13, orabout pH 14 may be used. Additionally, any alkali that can bring asolution pH to a range of about pH 12 to about pH 13, from about pH 12to about pH 14, and from about pH 13 to about pH 14 can be used.Suitable concentrations of alkali include from about 1.0 N to about 2.0N, from about 1.25 N to about 1.75 N, and from about 1.25 N to about 1.5N, and about 1.5 N as well as any number within the recited ranges.Without being limited, suitable alkali include NaOH and KOH.

In one example, approximately one volume of the sample suspended in adetergent-based collection medium can be treated with about one-halfvolume of 1.75 N NaOH solution. For example, in certain aspectsapproximately a 50 μl aliquot is removed from a sample suspended in adetergent-based collection medium and approximately 25 μl of 1.75 N NaOHsolution is added to the 50 μl aliquot sample. At room temperature, thesample treated with the denaturation reagent can be mixed by hand mixingor mechanical shaking at about 800 rpm, about 900 rpm, about 1000 rpm,between about 600 and about 1000 rpm, or between about 600 and 1200 rpm.The pH of the sample after addition of denaturation reagent can be about14. In another aspect, the pH can be about pH 12 or pH 13. Such basic pHwill both nick and denature a majority of the nucleic acid in thespecimen. In addition, alkaline treatment can disrupt interactionsbetween peptides and nucleic acids to improve accessibility of thetarget nucleic acid and degrade protein.

Alkaline treatment of protein effectively homogenizes the specimen toensure reproducibility of analysis results for a given sample. It canalso reduce the viscosity of the sample to increase kinetics, homogenizethe sample, and reduce background by destroying any endogenous singlestranded RNA nucleic acids, DNA-RNA hybrids or RNA-RNA hybrids in thesample. It also helps inactivate enzymes such as RNases and DNases thatmay be present in the sample. One skilled in that art would appreciatethat if RNA is the target nucleic acid (as opposed to DNA), differentreagents may be preferable including, but not limited to phenolextraction and TCA/acetone precipitation, and guanidiniumthiocyanate-phenol-chloroform extraction.

Other methods of denaturation may be employed such as utilizing aheating step, for example, heating the sample to about 95° C. toseparate the strands of nucleic acid. Enzymes such as helicase may beused as well.

In one aspect, 1.5 N to 2.0 N NaOH is added to the sample and heated. Inanother aspect, 1.75 N NaOH is added to the sample and heated. Thesample with denaturation reagent may be heated to about 60° C. to about80° C. for about 30 minutes, to about 65° C. to about 75° C. for about30 minutes, to about 67° C. to about 70° C. for about 30 minutes, or toabout 70° C. for about 30 minutes, or any number within the recitedranges. In another aspect, the sample with denaturation reagent isheated to about 60° C. to about 80° C. for about 20 to about 40 minutes,or to about 65° C. to about 75° C. for about 20 to about 40 minutes, toabout 67° C. to about 70° C. for about 20 to about 40 minutes, or toabout 70° C. for about 20 to about 40 minutes, or any number within therecited ranges. The goal of the described time and temperatureconditions is to provide for maximal denaturation of the sample in aminimum amount of time, while leaving the target nucleic acid in asuitable condition for carrying out the remaining steps ofhybridization, capture, washing, and detection. Therefore, the samplemay be heated in denaturation reagent for about 5 to about 120 minutes,about 10 to about 60 minutes, about 20 minutes to about 40 minutes,about 30 minutes, or any number within the recited ranges. It will bereadily understood by one of ordinary skill in the art that longerperiods of incubation at lower temperatures, or shorter periods ofincubation at higher temperatures, may be balanced to provide a similareffect to the conditions described herein.

Hybridization and Binding of Probes

After the sample containing the nucleic acid is denatured, it iscontacted with one or more polynucleotide probes under a conditionsufficient for the one or more polynucleotide probes to hybridize to thetarget nucleic acid in the sample to form a double-stranded nucleic acidhybrid. The probe can be full length, truncated, or synthetic DNA orfull length, truncated, or synthetic RNA. If the target nucleic acid isDNA, then the probe may be RNA and if the target nucleic acid is RNA,then the probe may be DNA. Preferably, the one or more polynucleotideprobes are diluted in a probe diluent that also can act as aneutralizing hybridization buffer (to neutralize the basic denaturationreagent).

The probe diluent used for DNA or RNA probes will differ due to thedifferent requirements necessary for DNA versus RNA stability. Forexample, if the probes are RNA, it is preferable to neutralize thesample first and than add the probe or alternatively, add the RNA probeand neutralizing agent (probe diluent) to the sample at the same time asNaOH can destroy RNA. The probe diluent can be used to dissolve anddilute the probe and also help restore the sample to about a neutral pH,e.g., about pH 6 to about pH 9, to provide a more favorable environmentfor hybridization. Sufficient volume of probe diluent, preferablyone-half volume of the sample, may be used to neutralize thebase-treated sample.

In an aspect, the probe diluent comprises a buffer, polyacrylic acid,NaOH and sodium azide. The probe diluent may comprise acetic acid. Inone aspect, the probe diluent comprises 2.2 M BES(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), 2.6% polyacrylicacid (PAA), 0.7 N NaOH and 0.05% sodium azide. The probe diluent maycontain from about 1.2 M to about 2.6 M BES, from about 1.5 M to about2.5 M BES; from about 1.75 M to about 2.25 M BES; from about 2 M to 2.4M BES, or about 2.2 M BES, as well as any number within the recitedranges. In one aspect the probe diluent may contain from about 2% toabout 3.0% PAA or, as well as any number within the recited ranges. Inanother aspect, the PAA concentration is from about 2.2% to about 2.7%.In yet another aspect, the PAA concentration is about 2.6%. In a furtheraspect the probe diluent may contain from about 0.6 N to about 0.8 NNaOH, for example, about 0.7 N NaOH. The concentration of NaOH generallyincreases as the amount of BES increases.

For full length probes, a heated alkaline solution may be added to thesample, then probe diluent may be added to the sample at roomtemperature, and then the sample may be reheated. Such a process caninhibit secondary structure from forming. Antibodies tend toirreversibly bind to structures with secondary structure. When usingnon-full length probes such as truncated or synthetic probes, heatingthe solutions or sample may not be necessary because secondarystructures issues are not present. In an aspect, the sample is notheated when used with truncated or synthetic probes.

After treatment with the denaturation reagent, an aliquot ofneutralization buffer, in an aspect the probe diluent described, inwhich the one or more probes are dissolved, can be added to the sampleunder appropriate conditions to allow hybridization or binding of theprobe and the target nucleic acid to occur. The neutralization buffermay contain a single buffering salt. In an aspect, the neutralizationbuffer does not contain more than a single buffering salt. Thehybridization condition is sufficient to allow the one or morepolynucleotide probes to anneal to a corresponding complementary nucleicacid sequence, if present, in the sample to form a double-strandednucleic acid hybrid.

Hybridization conditions suitable for the particular probes and diluentsdescribed herein are employed. For example, the probes and samplenucleic acids can be incubated for a hybridization time, preferably atleast about 5 to about 30 minutes, about 5 to about 20 minutes, or fromabout 7 to about 15 minutes, or about 10 minutes, as well as any numberwithin the recited ranges sufficient to allow the one or morepolynucleotide probes to anneal to a corresponding complementary nucleicacid sequence. The hybridization condition can include a hybridizationtemperature of at least about 65° C., about 68.5° C., and about 67° C.to about 70° C., as well as any number within the recited ranges. For agiven target nucleic acid and a given probe, one of ordinary skill inthe art can readily determine desired hybridization conditions byroutine experimentation. One of ordinary skill in the art will furtherappreciate that the time and temperature of hybridization must beoptimized, one with respect to the other. Thus, higher hybridizationtemperatures may be carried out for shorter periods of time and viceversa. Without being limited, stringent hybridization conditions may becontrolled by increasing the temperature, increasing the ionicconditions to above 0.5 M (for example, NaCl), or reducing theconcentration of PAA. As a non-limiting example, stringent hybridizationconditions may include performing a hybridization reaction at elevatedtemperatures, such as of at least about 65° C., at least about 68.5° C.,between about 67° C. to about 70° C. , and between about 69° C. to about70° C. Stringent hybridization conditions may also include elevatedtemperatures, such as of at least about 65° C., at least about 68.5° C.,and between about 67° C. to about 70° C.

In a non-limiting aspect, the probe is capable of hybridizing or bindingto nucleic acid molecules at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 98%, at least 99%, or 100% identical to nucleic acid moleculesassociated with HPV, genetic variants of HPV, HPV DNA of a high risk HPVtype, or HPV RNA of a high risk HPV type, or any one of high risk HPVtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 orany one of low risk HPV types 6, 11, 40, 43, 53, 61, 67, 69, 70, 71, 72,81, and 83. In another aspect, the probe is complementary to HPV,genetic variants of HPV, HPV DNA of a high risk HPV type, HPV RNA of ahigh risk HPV type, or any one of high risk HPV types 16, 18, 31, 33,35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or any one of low riskHPV types 6, 11, 40, 43, 53, 61, 67, 69, 70, 71, 72, 81, and 83.

In one aspect, the sample is suspended in detergent-based collectionmedium, the target nucleic acid is denatured with a denaturationreagent, and hybridized to nucleic acid probes suspended in aneutralizing buffer. In another aspect the neutralizing buffer is theprobe diluent of the present invention. The probe diluent can comprises2.2 M BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), 2.6%polyacrylic acid, 0.7 N NaOH and 0.05% sodium azide.

Capture

After the probes are allowed to hybridize to the target nucleic acidmolecule and to form a double-stranded nucleic acid hybrid, the hybridis captured by a molecule that is specific for the double-strandednucleic acid hybrid. Molecules specific for the double stranded nucleicacid hybrids include, but are not limited to, monoclonal antibodies,polyclonal antibodies, proteins such as but not limited to RNAse H,nucleic acids including but not limited to aptamers, or sequencespecific nucleic acids. Aptamers are short stretches of random sequencesthat are successively selected from a library of sequences byhybridizing to a target, amplifying the hybridized aptamers, andrepeating the selection process. In one aspect the molecule specific forthe double stranded nucleic acid hybrid is captured by an antibody,known as an anti-hybrid antibody.

In one aspect, a first anti-hybrid antibody is immobilized onto asupport using techniques that are standard in the art. Examples ofsuitable supports include covalent linkages or adsorption, for example,protein-protein interactions, protein-G beads, biotin-streptavidininteraction, EDAC to link to a carboxyl or tosyl group, etc., orhybridization directly onto the solid support using, for example,sequence specific nucleic acids in an affinity column.

Supports include but are not limited to beads, magnetic beads, which asindicated previously include paramagnetic, diamagnetic, ferromagnetic,ferromagnetic, and diamagnetic beads, columns, plates, filter paper,polydimethylsiloxane (PDMS), and dipsticks. Any support can be used aslong as it allows extraction of the liquid phase and provides theability to separate out bound and unbound antibodies. Magnetic beads areparticularly useful in that they can be left in the solution and theliquid phase can be extracted or decanted, if a magnetic field isapplied to immobilize the beads. Beads that are small and have a highsurface area are preferable, such as beads about 1 μm in diameter. Otherbeads that employ charge switching or silica capture (as opposed tomagnetic fields) may be used as well.

The hybrids are incubated with the anti-hybrid antibody attached to thesupport for a sufficient amount of time to allow capture of thedouble-stranded nucleic acid hybrids by the immobilized anti-hybridantibodies. In an aspect, the support is a bead.

The anti-hybrid antibody may be monoclonal or polyclonal. In one aspectthe antibody is monoclonal. In one aspect, the antibody is coupled tosupport by an 1-ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride (EDAC) linker. In one aspect, the support is a polystyrenebead. In an aspect, the support or bead coupled to the antibody isdiluted in a bead dilution buffer. The bead dilution buffer is helpfulin minimizing protein denaturation on the bead. One example of a beaddilution buffer comprises 6% casein, 100 mM Tris-HCl, 300 mM NaCl, and0.05% sodium azide.

In an aspect, the beads coated with the anti-hybrid antibody areincubated with the sample at about 67° C. to about 70° C. for about 30minutes. In another aspect, the beads and sample are incubated at about68° C. to about 69° C. for about 30 minutes. In yet another aspect, thebeads and sample are incubated at about 68.5° C. for 30 minutes. Theincubation time can range from about 5 minutes to about 60 minutes, fromabout 15 minutes to about 45 minutes, from about 20 minutes to about 40minutes, or any number within the recited ranges, and is generallyinversely proportional to the temperature. It will be understood bythose skilled in the art that the incubation time, temperature and/orshaking conditions can be varied to achieve alternative capture kineticsas desired.

Following capture of the target nucleic acid/probe hybrid as describedabove, the captured hybrid may be separated from the rest of the sampleby washing away of non-captured nucleic acids.

Conjugation

Another step in the method can involve providing a second antibody thatis also specific for double stranded nucleic acids hybrids oralternatively is specific for the first antibody. The second antibodymay be detectably labeled, either directly or indirectly, and may be amonoclonal or polyclonal antibody. In an aspect, the second antibody ismonoclonal. In another aspect, the second antibody is directly labeledwith a detectable marker and is monoclonal. The second antibody is usedto detect the presence of double-stranded nucleic acid hybrids. In oneaspect, the second antibody has a label that must react with a substrateto provide a signal that can be detected. The second antibody may bedissolved in a suitable buffer. In one aspect the buffer comprises 100mM TrisHCl, pH 7.4, 0.5 M NaCl, 0.1 mM ZnCl₂, 1.0 mM MgCl₂, 0.25% Tween20, 0.2 mg/ml RNase A, 4% hydroxypropyl-b-cyclodextrin (cyclodextrin),30% bead dilution buffer as discussed previously, 0.05% goat IgG, 0.05%sodium azide.

In an aspect, the conjugation reaction takes place at room temperature.In an aspect, the conjugation reaction takes place at room temperaturefor between about 1 hour and 2 hours. In another aspect, the conjugationreaction takes place at room temperature for about 2 hours. In anotheraspect the conjugation reaction takes place at about 37° C., about 45°C., or about 50° C. In an aspect the conjugation reaction takes place atabout 37° C., about 45° C., or about 50° C., between 35° C. and about40° C., between 40° C. and about 50° C. for between about 20 minutes and40 minutes. In an aspect the conjugation reaction takes place at about37° C., about 45° C., or about 50° C. for between about 20 minutes and40 minutes. In another aspect the conjugation reaction takes place atabout 45° C. for about 30 minutes.

It will be understood by those skilled in the art that any detectablelabel such as, but not limited to, an enzyme, radioactive molecule,fluorescent molecule, or metal particle such as gold particle can beused. In certain aspects, the detectable label is alkaline phosphatase.Methods of conjugating a label to an antibody are known. For example, anantibody can be reduced with dithiothreitol (DTT) to yield monovalentantibody fragments. The reduced antibody can then be directly conjugatedto maleinated alkaline phosphatase by the methods of Ishikawa et al., J.Immunoassay 4:209-237 (1983) and Means et al., Chem. 1: 2-12 (1990), thecontents of each of which are incorporated herein by reference in itsentirety, and the resulting conjugate can be purified by HPLC. Theconjugate may also be purified using any type of size-exclusionchromatography. One benefit of purification is that the conjugates ofone protein to one antibody can be separated from those conjugates withother ratios of protein to antibody.

In another aspect, the double-stranded nucleic acid hybrids can bedetected with a second anti-hybrid antibody that is not directlylabeled. For example, the second antibody can be a mouse immunoglobulinthat is detected by a labeled goat anti-mouse antibody.

Wash

Following conjugation with the second antibody, the sample is washedwith a based wash buffer. The wash buffer may contain one or moredetergents or may be free of a detergent. If the wash buffer contains adetergent, the detergent may be an ionic or a non-ionic detergent. Oneexample of a non-ionic detergent is Triton-X. The detergent may bepresent in the wash buffer at a concentration of about 0.05% to about1.5%, or from about 0.075% to about 1.0%, or from about 0.1% to about0.75%, or about 0.5% or any number within the recited ranges. Oneexample of a suitable wash buffer comprises 40 mM Tris, pH 8.2, 100 mMNaCl, 0.5% Triton-X 100 and 0.05% sodium azide.

The sample may be washed with the wash buffer from one to ten times, orfrom three to seven times, or from four to six times, or five times, orany number within the recited ranges. The sample may also be washed witha single wash buffer or with multiple wash buffers. Each wash may usethe same wash buffer or a different wash buffer. For example, adetergent-containing wash buffer may be used for one wash while adetergent-free wash buffer may be used for another wash. In an aspect,one of the wash buffers does not include Triton.

One benefit of the detergent-containing wash buffer is the positiveeffects on bead behavior when compared to detergent-free wash buffers.The detergent-containing wash buffer allows for rapid, efficient, andresilient binding of the beads to the magnetic field. Binding of thebeads to the magnetic field is strong enough that beads remain boundthrough physical inversion and decanting. While detergent-free washbuffers generally do not allow for physical inversion without bead loss,they may be used for other purposes. One example of the use of adetergent-free wash buffer is to remove or dilute a detergent in thesample thereby reducing any likely detection problems.

Detection

The label present on the second, or third, or more, antibody is detectedto thus indicate the presence of the target nucleic acid molecule.Methods for detecting various labels are known in the art. For example,colorimetry, radioactive, surface plasmon resonance, orchemiluminescence methods are described by e.g., Coutlee et al., J.Clin. Microbiol. 27:1002-1007 (1989), the contents of which areincorporated herein by reference in its entirety.

For example, a bound alkaline phosphatase conjugate can be detected bychemiluminescence with a reagent such as a LUMI-PHOS 530 reagent(Lumigen, Detroit, Mich.) or DR2 (Applied Biosystems, Foster City,Calif.) using a detector such as an E/LUMINA luminometer (SourceScientific Systems, Inc., Garden Grove, Calif.), an OPTOCOMP ILuminometer (MGM Instruments, Hamden, Conn.), or the like, such as aVeritas Microplate Luminometer by Turner Biosystems. Multiple detectiontechniques can also be used in sequence or in parallel. For example, theconjugate may be detected by chemiluminescence and fluorescence. Inanother aspect, the conjugate can be detected by chemiluminescence.

Detectors using different detection techniques for the conjugate may bereversible or irreversibly attached, for example in a modular fashion,to a machine that is capable of performing the method for determiningthe presence of a target nucleic acid molecule in a sample.

As described herein, detection of the label on the second antibody isindicative of the presence of one or more of the target nucleic acidmolecules in the sample that are complementary to the one or moreprobes. Following washing, the sample is suspended in a detection bufferthat for example, contains the substrate for the label on the secondantibody.

In one aspect, the sample is comprised of cervical cells. The method fordetermining the presence of a target nucleic acid molecule in a sampleof cervical cells comprises suspending the sample in a detergent-basedcollection medium and mixing by hand mixing. In another aspect themixing is mechanical. An approximately 50 μl aliquot of the sample isremoved and mixed with about 25 μl of a denaturation reagent. The sampleis mixed by hand mixing or mechanical shaking at between about 600 toabout 1200 rpm for about 30 to about 60 seconds and heated at about 70°C. for about 30 minutes. High risk HPV RNA probes are prepared in adiluent and diluted to about 375 ng/ml. About 40 μl of diluted probe isadded to the sample on a 70° C. heating block. The samples are furtherincubated at approximately 68.5° C. with shaking at about 1150 rpm forabout 30 minutes. The supernatant can be removed by a dropper bottle orother low tech device. About 35 μl of the detection reagent is added tothe sample. The detection reagent contains a second antibody that islabeled. The second antibody is specific for double-stranded nucleicacid hybrids. The sample containing the detection reagent is incubatedat about 45° C. for about 30 minutes, placed on a magnetic rack forabout 30 seconds to 3 minutes and the supernatant is decanted. Inanother aspect the sample containing the detection reagent is incubatedat room temperature. The sample is then washed with wash buffer aboutfour or five times.

Anti-hybrid Antibodies

The double-stranded nucleic acid hybrids formed in accordance with thepresent invention can be captured and detected using antibodies that arespecific to double-stranded nucleic acid hybrids. The antibody isspecific to double-stranded hybrids, such as but not limited to RNA-DNA;DNA-DNA; RNA-RNA; and mimics thereof, where mimics refer to moleculesthat behave similarly to RNA-DNA, DNA-DNA, or RNA-RNA hybrids. Theanti-double-stranded nucleic acid hybrid antibody, i.e., the anti-hybridantibody that is utilized will depend on the type of double-strandednucleic acid hybrid formed. In one aspect, the anti-hybrid antibody isimmunospecific to RNA-DNA hybrids.

It will be understood by those skilled in the art that either polyclonalor monoclonal anti-hybrid antibodies can be used and/or coupled to beadsand/or immobilized on a support in the present assay as described below.Monoclonal antibody prepared using standard techniques can be used inplace of the polyclonal antibodies. Monoclonal antibodies may beproduced by methods that are standard in the art. In an aspect, theantibodies used for capture and detection of the target nucleic acid aremonoclonal antibodies. In an aspect, monoclonal antibodies support highstringency incubation temperatures during the capture step. Withoutbeing limited, the high stringency incubation temperatures during thecapture step may be between about 65° to about 75° C. or between about68° to about 75° C. The first and second antibodies may be the same forcapture and detection (i.e., produced by the same hybrid myeloma cellline) or may be different and produced by different hybrid myeloma celllines. In one aspect, the first and second monoclonal antibodies usedfor capture and/or detection are the same and are specific for RNA-DNAhybrids. Also included are immunofragments or derivatives of antibodiesspecific for double-stranded hybrids, where such fragments orderivatives contain binding regions of the antibody.

For example, a monoclonal anti-RNA-DNA hybrid antibody derived frommyeloma cells fused to spleen cells that are immunized with an RNA-DNAhybrid can be used. The hybrid-specific antibody can be purified byaffinity purification against RNA-DNA hybrids immobilized on a solidsupport, for example as described in Kitawaga et al., Mol. Immunology,19:413 (1982); and U.S. Pat. No. 4,732, 847, the contents of each ofwhich are incorporated herein by reference in their entirety.

Other suitable methods of producing or isolating antibodies, includinghuman or artificial antibodies, can be used, including, for example,methods that select recombinant antibody (e.g., single chain F_(v) orF_(ab), or other fragments thereof) from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing arepertoire of human antibodies (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); and U.S. Pat. No. 5,545,806 and U.S. Pat. No. 5,545, 807,the contents of each of which are incorporated herein by reference intheir entirety).

In one aspect, the target nucleic acid to be detected is DNA (e.g., HPVgenomic DNA or cDNA) or RNA (e.g., mRNA, ribosomal RNA, nuclear RNA,transfer RNA, viral RNA, heterogeneous nuclear RNA), wherein the one ormore polynucleotide probes are polyribonucleotides orpolydeoxyribonucleotides, respectively. In a preferred aspect, thedouble-stranded nucleic acid hybrids are DNA-RNA hybrids formed byhybridization of target DNA and probe RNA, and can be detected using anantibody that is immunospecific to RNA-DNA hybrids.

In an aspect of the present invention, a monoclonal anti-RNA-DNA hybridantibody derived from a hybridoma cell line is used. Such hybridoma celllines are described in U.S. Pat. No. 4,865,980, U.S. Pat. No. 4,732,847,and U.S. Pat. No. 4,743,535, the contents of each of which areincorporated herein by reference in their entirety. Hybrid-specificmonoclonal antibodies may be prepared using techniques that are standardin the art. The hybrid-specific monoclonal antibody may be used for bothcapturing and detecting the target nucleic acid.

While any vertebrate may be used for the preparation of polyclonalanti-RNA-DNA hybrid antibodies, goats or rabbits are preferred.Preferably, a goat or rabbit is immunized with a syntheticpoly(A)-poly(dT) hybrid by injecting the hybrid into the animal inaccordance with conventional injection procedures. Polyclonal antibodiesmay be collected and purified from the blood of the animal withantibodies specific for the species of the immunized animal inaccordance with well-known antibody isolation techniques. For theproduction of monoclonal antibodies, the spleen can be removed from theanimal after a sufficient amount of time, and splenocytes can be fusedwith the appropriate myeloma cells to produce hybridomas. Hybridomas canthen be screened for the ability to secrete the anti-hybrid antibody.Selected hybridomas may then be used for injection into the peritonealcavity of a second animal for production of ascites fluid, which may beextracted and used as an enriched source of the desired monoclonalantibodies incorporated herein by reference.

Polynucleotide Probes

The polynucleotide probes are designed to hybridize or bind with thetarget nucleic acid molecules. In another aspect, the polynucleotideprobes are designed to bind to target nucleic acid molecules. In oneaspect, the probes are capable of hybridizing or binding to HPV and HPVhigh risk variants. In an additional aspect, the polynucleotide probesare specific for HPV and HPV high risk variants. High risk (HR) nucleicacid probes can include probes for HPV high risk types 16, 18, 31, 33,35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 82. In other aspects the RNAor DNA probes are fragments. In an aspect, the probes are about 6 toabout 8 kilobases in length, preferably about 7.5 kilobases, and may beproduced using a plasmid template using a BLUESCRIPT vector. However,other plasmids, vectors and methods are known in the art and could alsobe used to produce the RNA probes described herein.

The probes may vary in amount from about 7.5 ng to about 60 ng per HPVtype per assay, or from about 20 ng to about 45 ng per HPV type perassay, or about 30 ng of probe for each HPV type per assay is used.Thus, in one aspect the HR probes consist of or consist essentially ofone or more probes for HPV high risk types 16, 18, 31, 33, 35, 39, 45,51, 52, 56, 58, 59, 66, 68, and 82 or low risk HPV types 6, 11, 40, 43,53, 61, 67, 69, 70, 71, 72, 81, and 83, wherein about 30 ng of eachprobe is used per assay for detection of the target nucleic acidmolecule.

The RNA probes may be short synthetic RNA probes that specifically bindonly to the target nucleic acid molecule. Examples are described in U.S.patent application Ser. No. 12/426,076, filed on Apr. 17, 2009, thecontents of which are incorporated herein by reference in its entirety.

Cross-Reactivity

The present invention also provides for assay compositions, probes, andconditions wherein cross-reactivity between HPV HR probe sets and lowrisk HPV types is dramatically reduced when compared to the standard FDAapproved HPV assay and probe set. In one aspect, the HPV HR probe set isselected from the group consisting of HPV high risk types 16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or low risk HPV types6, 11, 40, 43, 53, 61, 67, 69, 70, 71, 72, 81, and 83. Using the presentassay with these HR HPV probes, cross-reactivity between low risk HPVtypes and high risk HPV probes is reduced. See, for example, U.S. patentapplication Ser. No. 12/426,076.

The present invention also provides a method for determining thepresence of a target nucleic acid molecule, such as HPV, in a sample inabout 2 hours or less, about 2.5 hours or less, about 3 hours or less,about 3.5 hours or less, about 4 hours or less, about 5 hours or less,about 6 hours or less, about 7 hours or less, about 8 hours or less,about 12 hours or less, about 24 hours or less, in other aspects, lessthan about 3.5 hours for at least 10 samples using the methods discussedabove. One reason why the presence of HPV or other target nucleic acidmolecules can be determined in short periods of time is because themethod does not amplify the target nucleic acid molecule prior todetection. Instead of target amplification, signal amplification may beused to accurately detect the presence of HPV or other target nucleicacid molecules. In an aspect, the methods of the disclosure may includea signal amplification step. In an aspect, the methods of the disclosuredo not include a target amplification step. In another aspect, themethods of the disclosure may include a signal amplification step and notarget amplification step.

The present disclosure also provides methods and assays for detectingcancer, for example cervical cancer, by detecting the presence of atarget nucleic acid molecule, such as HPV, in a sample in about 2 hoursor less, about 2.5 hours or less, about 3 hours or less, about 3.5 hoursor less, about 4 hours or less, about 5 hours or less, about 6 hours orless, about 7 hours or less, about 8 hours or less, about 12 hours orless, about 24 hours or less, in other aspects, less than about 3.5hours for at least 10 samples using the methods and assays discussedabove.

It will be understood to those skilled in the art that the presentinvention can be carried out on a number of platforms including, but notlimited to, tubes, dipsticks, microarrays, microplates, 384 well plates,other microtiter plates and microfluidic systems. It will be understoodto those skilled in the art that the present, as relevant to developingcountries, can utilize low technology methods such as dropper bottles,rubber bulbs, Pasteur pipettes, or squirt bottles for steps involvingmovement of liquid. These devices deliver relatively precise volumeswithin the approximate ranges that are needed for the assay. In anaspect, the methods of the disclosure do not include automatic pipettorsor other battery powered or energy powered pipetting devices.

Another aspect of the present invention provides a collection mediuminto which samples containing the target nucleic acid are collected. Thecollection medium provides sample stability for several days, severalweeks, or several months. For example, the collection medium may providesample stability for at least 1 week, at least 2 weeks, at least 3weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3months, at least 4 months, at least 5 months, at least 6 months, fromabout 1 week to about 4 weeks, from about 1 month to about 3 months,from about 3 to about 4 months, or from about 3 month to 6 months. Inanother aspect, the collection medium provides sample stability for atleast 21 days at 33° C. or at least 6 months at 20° C. In an aspect theabove sample is a cervical cell sample or a human cervical cell sample.Suitable collection media are described herein. In one aspect, thecollection medium comprises, consists of, or consists essentially ofNP-40, deoxycholate, Tris-HCl, EDTA, NaCl, and sodium azide. In otheraspects, the collection medium comprises, consists of, or consistsessentially of 1.0% NP-40, 0.25% sodium deoxycholate, 50 mM Tris-HCl, 25mM EDTA, 150 mM NaCl, and 0.05% sodium azide.

Another aspect is a detergent-containing wash buffer comprising,consisting of, or consisting essentially of 40 mM Tris pH 8.2, 100 mMNaCl, 0.1% to 0.5% Triton X-100, and 0.05% sodium azide. Yet anotheraspect is a detergent-free wash buffer comprising, consisting of, orconsisting essentially of 40 mM Tris pH 8.2, 100 mM NaCl, and 0.05%sodium azide.

Sample Conversion for Recovery, Detection, and Analysis of Nucleic AcidMolecules

An aspect relates to adding a collection medium to a sample which hasbeen previously prepared for diagnostic analysis. In one aspect, thesample to which the collection medium is added has been previouslyprepared using a liquid based cytology (LBC) assay. LBC media cancontain tissue fixatives such as alcohol and formalin which serve tostabilize the sample, inhibit bacterial growth, preserve cell morphologyand diagnostic clusters, and assure the preparation of a tissuemonolayer cytology slides. However, many compositions used to preservebiological samples, such as SUREPATH, contain alcohol or formalin whichcan be detrimental to analyzing nucleic acid molecules. In an aspect,the cytology slides contain cervical cell samples or any otherbiological sample capable of being evaluated. In an aspect, the SUREPATHmedia is used to prepare LBC sample.

In addition to cytology preparation, LBC samples can be used fordetection of disorders, such as common sexually transmitted pathogens,including Human Papillomavirus (HPV), Neisseria gonorrhoeae (GC), andChlamydia trachomatis (CT), among others. As a supplement to itsapplication as a screening tool, LBC samples can be used to monitorpatients' viral clearance after treatment for a particular disease,informing further follow-up and treatment regimens. In an aspect, HPVtesting of LBC samples can be used to monitor patients' viral clearanceafter treatment for a cervical-based disease.

In an aspect, a biological sample is collected and preserved in a media,such as the SUREPATH media. The preserved media containing thebiological sample is stored until further processing is required. Thepreserved media containing the biological sample can be removed andsuspended in water thereby forming a “soft pellet.” A portion of thesoft pellet may be removed and analyzed on a slide. In an aspect, thesample is prepared using a LCB assay. Instead of adding morepreservation media, such as SUREPATH, to the remaining soft pelletsuspension, the detergent-based collection media described herein may beadded to the remaining biological sample. This is advantageous in that asample dispersed in the detergent-based collection media describedherein can be directly analyzed in a nucleic acid molecule detectionassay. Additionally, the biological sample suspended in thedetergent-based collection medium is stable for at least 11 days at roomtemperature (FIG. 6).

Any of the disclosed detergent-based collection media are capable ofbeing added to the soft pellet. In another aspect, a detergent andchelator media may be used to resolubilize the pellet. In a non-limitingaspect, a collection media including about 0.5% to about 2.0% NP-40,about 0.10% to about 0.40% sodium deoxycholate, about 25 mM to about 75mM Tris-HC1, about 10 mM to about 50 mM EDTA, about 50 mM to about 200mM NaCl, and about 0.01% to about 0.10% sodium azide can be used tosolubilize the soft pellet. After the addition of the detergent-basedcollection media, the soft pellet sample may be analyzed in conjunctionwith any of the methods or assays described herein.

Kit

Also provided is a kit for the detection of a target nucleic acidmolecule in a sample, the kit comprising, consisting of or, orconsisting essentially of:

-   -   a) a collection medium;    -   b) a denaturation reagent;    -   c) at least one polynucleotide probe;    -   d) a bead coated with a first anti-hybrid antibody;    -   e) a detection reagent comprising a second anti-poly hybrid        antibody, wherein the second antibody is detectably labeled;    -   f) a wash buffer; and    -   g) a second detection reagent comprising a substrate for the        label on the second antibody.

The collection medium, denaturation reagent, bead, first and secondantibodies, polynucleotide probes, detection reagents, and wash buffershave been previously described.

The kit may also include instructions for describing proceduresassociated with the disclosed methods and assays. The kit may alsoinclude a means for transcribing patient information. In an aspect, themeans includes paper, a computer, or a device capable of transmittingpatient information. The kit can include all the necessary components tocomplete the methods at the same location where the patient sample istaken.

In an aspect, the kit may include color coded reagents associated withthe detection assay. The reagent vials are color coded for ease of useand can be included in a kit. The reagent bottles may also be identifiedby symbols, letters, or other known identifiers.

As the individual components of the kit come together in an easy to useplatform, one advantage of the kit described herein is that it providesfor immediate testing o samples. This allows for rapid determination ofpatient results.

In an aspect, methods of the disclosure can include the collection andprocessing of patient samples in the field. In one aspect, after thesamples are collected, some of the method steps are conducted at thesame location where the patient samples are collected. In anotheraspect, all of the method steps can be conducted at the same locationwhere the samples are collected. The location may be a village, clinic,laboratory, or communal area where individuals receive medical checkupsand evaluations. The location may be permanent or temporary. In anaspect, the nucleic acid molecule is detected at a location, such as alaboratory or clinic, which is different from where the samples aretaken. In an aspect, the kit is designed for use in a developing countryor geographical areas where access to medical care is not readilyavailable.

System

In an aspect, the sample may be analyzed using a portable system fordetecting the presence of a target nucleic molecule acid in a sample.The portable system may include a heater configured for heating multiplesamples; a luminometer configured to detect the chemiluminescence of atarget nucleic acid molecule on a support coated with a first antibodyand conjugated to a second antibody labeled with a detectable marker;and a monitor configured to report chemiluminescence data (FIG. 7). Inan aspect, the heater is a combination heater/shaker. In an aspect, thesystem is configured to simultaneously analyze 90 samples at a timetogether with 6 controls (96 samples altogether). In an aspect, both theluminometer and the heater are configured to simultaneously analyze 90samples at a time together with 6 controls (96 samples altogether). Thesystem is also capable of reporting the results of at least 500 samplesat a time. The portable system and associated reagents are set forth inFIGS. 6, 7, and 8.

Without being limited, the individual components of the portable systemcan be easily transported by an individual, such as a doctor orlaboratory technician, and weighs less than 10 pounds, less than 20pounds, less than 30 pounds, or less than 40 pounds. In another aspect,the entire portable system for detecting target nucleic acid moleculesweighs less than 20 pounds, less than 30 pounds, less than 40 pounds, orless than 50 pounds, or less than 100 pounds and is designed for easytransportation.

The portable system and assay can be designed for use in the field orlocation where samples are collected and require a small footprint ofbench-top work space (for example, about 25×50 cm or less). In anaspect, the portable system is configured to work without electricity,mains, or running water. The portable system may also run entirely onbatteries. The portable system can be designed for use with thedescribed kit, assays, with the methods described herein. Together, thekit and portable system provide an efficient and rapid way to analyzesamples and detect nucleic acid samples in developing countries or otherareas which may lack sophisticated laboratory equipment or facilitiesfor analyzing laboratory samples.

It is noted that when ranges are described herein, any number withinthat range is contemplated in the inventions described herein. Theentire contents of all patents, patent applications, scientificpublications, etc. listed are incorporated in their entirety byreference. The examples below are not meant to limit the scope of theinvention.

EXAMPLES Example 1 Assay Using Cervical Samples and HPV probes

A total of 324 physician collected cervical samples were collected in adetergent based collection medium and tested for the presence ofhigh-risk HPV.

A 1 ml sample was vortexed to homogenize the sample and a 50 μl aliquotwas removed and combined with 25 μl of denaturation reagent (1.75 NNaOH) in the assay microplate. This was shaken to mix and incubated at70° C. for 30 minutes to create single stranded DNA. To this, 40 μl of aneutralization buffer (probe diluent −2.2M BES, 2.6% PAA, 0.7 N NaOH and0.05% sodium azide) containing RNA probes for 16 HPV types was added tocreate a neutral pH and incubated at 68.5° C. for 10 minutes.

Following this, 10 μl of antibody conjugated paramagnetic beads(approximately 1 μm carboxylated SERADYN beads from Thermo Fisher) wereadded to the reaction and incubated for an additional 30 minutes at68.5° C. The RNA probes and DNA target molecules that were complementaryto each other bind and create RNA-DNA hybrids. The hybrids then capturedby a RNA-DNA hybrid specific antibody coated on the paramagnetic SERADYNbeads.

Following incubation, the paramagnetic beads are separated from theliquid phase/supernatant by exposure to a magnetic field. Thesupernatant waste is removed by decanting and 35 μl of detection reagent1 (secondary antibody conjugated enzyme comprising a monoclonalanti-RNA-DNA hybrid antibody conjugated to alkaline phosphatase) isadded and incubated at 45° C. for 30 minutes. The secondary antibodybinds the RNA-DNA hybrid-antibody-conjugated paramagnetic bead complex.Non-bound secondary antibody is washed away using a detergent based washbuffer (40 mM Tris, pH 8.2, 100 mM NaCl, 0.1% Triton-X 100 and 0.05%sodium azide).

A substrate (dioxetane-based substrate from ABI, called DCP Star, withEmerald II enhancer) is added to the washed beads and wells that containhigh-risk HPV DNA create light that is detectable by a luminometer andmeasured in RLUs (relative light units). An assay positive standardcontaining 1 μg/ml of HPV DNA is used to establish the positive cutoff.All sample RLU values are divided by the RLU value for the positivestandard creating a RLU/CO (RLU to cutoff value). Results are reportedin RLU/CO and anything greater than or equal to 1.0 is consideredpositive.

Example 2 Stability Testing

Following initial testing, samples were stored at room temperature and33° C. to observe the stability of the samples. Testing was conducted asfar as 21 days post collection. FIGS. 3 and 4 demonstrate that theRLU/CO value for each sample does not change with time up to 21 days. A2×2 analysis comparing baseline results to the results after 21 days ofstorage and scatter plot analysis demonstrated the linearity of theRLU/CO values with time. Based on these data, it is possible to concludethat samples collected and stored at either room temperature or 33° C.for as long as 21 days provide comparable RLU/CO values as tested atbaseline. Using linear mixed model comparison of RLU/CO values againstthe temperature of storage the P values are 0.8803 for room temperatureand 0.9517 for samples stored at 33° C. indicating that values areequal.

Example 3 Limit of Detection Studies

The limit of detection (LOD) of HPV 16 was measured by serial dilutionof HPV-16 plasmid target in the collection medium. The test resultsdemonstrate a S/N≧2.0 for a 0.2 μg/ml HPV 16 plasmid which is equivalentto 1000 copies of HPV 16 DNA. See FIG. 5. In addition, three-weekstability studies were performed on clinical specimens at elevatedtemperatures to mimic the conditions in an area subject to relativelyelevated temperatures, such as Rwanda. All biologically labile kitcomponents (RNA probe, capture antibody, detection antibody-enzymeconjugate, and substrate) were stabilized as part of the productionprocess and monitored for stability over 18 months at 37° C.

Example 4 SUREPATH Pellet Conversion and Recovery of Nucleic Acids

In this example, the typical workflow for SUREPATH pellet conversion andnucleic acid recovery is described. Workflow for SUREPATH media includesinitial collection of the primary sample, which can be cytobrushemployed for collection of cervical epithelium cells at a transitionzone or sample collection point. An average of 2×10^8 cervical cells arecollected per brush, which can be preserved in 10 mL of SUREPATH mediain a collection vial. The vial can be sealed and cells incubated in thefixative media at room temperature or 4° C. until further processing isperformed. The cell suspension can then be subjected to an automateddensity gradient purification scheme, and the resulting cellular pellet,with an average total cell number of 1.6×10¹^8 cells, can be suspendedin a final volume of 1 mL water. This water pellet can be referred to asa “soft pellet” or “undiluted soft pellet”.

200 uL of the soft pellet can be used in an automated slide preparationprotocol for cytology, leaving on average 1.3×10^8 total cells in an 800μL volume. After removal of the 200 aliquot for slide preparation, 1-2mL of fresh SUREPATH media can be added to the remaining 800 μL softpellet to stabilize and preserve the soft pellet in case the slide mustbe remade. In most cases, this remaining 2-3 mL sample is destroyedafter cytology results are reported.

The detergent-based medium described herein can be added to theremaining 800 μL soft pellet. Any of the detergent-based collectionmedium described herein can be added to the remaining 800 μL softpellet. In an aspect, the media may contain 1.0% NP-40, 0.25% sodiumdeoxycholate, 50 mM Tris-HCl, 25 mM EDTA, 150 mM NaCl and 0.05% sodiumazide. After the addition of the detergent-based collection media, thesoft pellet sample can be analyzed in conjunction with any of themethods or assays described herein.

What is claimed is:
 1. A composition comprising: (a) a biological samplesuspended in a collection medium, wherein said collection mediumcomprises NP-40, sodium deoxycholate, and EDTA; (b) a denaturationreagent; (c) at least one polynucleotide probe capable of binding to atarget nucleic acid molecule; (d) a support coated with a firstantibody; and (e) a second antibody labeled with a detectable marker. 2.The composition of claim 1, wherein said biological sample is a cervicalcell sample.
 3. The composition of claim 1, wherein said collectionmedium comprises about 0.8% to about 1.5% NP-40, about 0.20% to about0.40% sodium deoxycholate, and about 20 mM to about 40 mM EDTA.
 4. Thecomposition of claim 1, wherein a RLU/CO value for said biologicalsample does not change with time during at least a 21 day period whenstored in said collection medium at 33° C.
 5. The composition of claim1, wherein said at least one polynucleotide probe is selected from thegroup consisting of probes for HPV high risk types 16, 18, 26, 31, 33,35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and
 82. 6. The composition ofclaim 1, wherein said collection medium further comprises sodium azide.7. A composition comprising: (a) a biological sample suspended in acollection medium comprising NP-40, sodium deoxycholate, and EDTA; and(b) a polynucleotide probe.
 8. The composition of claim 7, wherein saidcollection medium further comprises sodium azide.
 9. The composition ofclaim 7, wherein said biological sample is a cervical cell sample. 10.The composition of claim 7, wherein a RLU/CO value for said biologicalsample does not change with time during at least a 21 day period whenstored in said collection medium at 33° C.
 11. The composition of claim7, wherein nucleic acid molecules in said biological sample aredenatured.
 12. The composition of claim 7, further comprising: (c) asupport coated with a first antibody.
 13. The composition of claim 12,further comprising: (d) a second antibody.
 14. The composition of claim13, wherein said second antibody is labeled with a detectable marker.15. A kit for the detection of a target nucleic acid molecule in asample, comprising, a) a collection medium comprising NP-40, sodiumdeoxycholate, and EDTA; b) a denaturation reagent; c) a support coatedwith a first anti-hybrid antibody; d) a detection reagent comprising asecond anti-poly hybrid antibody, wherein the second antibody isdetectably labeled; e) a detergent-based wash buffer; and f) a seconddetection reagent comprising a substrate for the label on the secondantibody.
 16. The kit of claim 15, wherein said collection mediumfurther comprises sodium azide.
 17. The kit of claim 15, wherein saiddetergent-based wash buffer comprises about 0.8% to about 1.5% NP-40,about 0.20% to about 0.40% sodium deoxycholate, about 30 mM to about 60mM Tris-HCl, about 20 mM to about 40 mM EDTA, about 100 mM to about 200mM NaCl, and sodium azide.
 18. The kit of claim 15, wherein saiddetergent-based wash buffer comprises about 40 mM Tris pH 8.2, 100 mMNaCl, 0.1% - 0.5% Triton x-100, and 0.05% sodium azide.
 19. The kit ofclaim 15, further comprising a probe diluent, wherein the diluentcomprises BES, polyacrylic acid, NaOH and sodium azide.
 20. The kit ofclaim 15, wherein the denaturation reagent is 1.75 N NaOH.
 21. The kitof claim 15, further comprising at least one polynucleotide probecapable of binding to human papillomavirus (HPV) and genetic variantsthereof.
 22. The kit of claim 21, wherein said at least onepolynucleotide probe is selected from the group consisting of probes forHPV high risk types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,66, 68,and
 82. 23. The kit of claim 15, further comprising an apparatusconfigured to detect the presence of a target nucleic acid in a sample.24. The kit of claim 23, wherein said system is a luminometer.